The dopamine transporter (DAT) is the primary target for the abused psychostimulants such as amphetamines (AMPHs) and cocaine. DAT belongs to the family of NeurotransmitterSodium: Symporters (NSS) and Is responsible for rapid clearance of dopamine from the extracellular space. It is our long term goal to understand the molecular and cellular processes governing the activity and availability of DAT in the presynaptic membrane and how these processes are altered in diseased states involving dysfunction of dopaminergic signaling. To perform its function, DAT participates in a network of protein-protein interactions and protein-llpid interactions that ensure proper trafficking, localization and catalytic activity of the transporter. We hypothesize that interaction ofthe DAT C-terminus with Ca2+/calmodulin dependent protein kinase Ila (CaMKIlo) and with PDZ (PSD-95/Discs-large/ZO-1 homology) domain proteins Is of central mportance for these processes. In knock-in mice expressing a DAT mutant incapable of forming PDZ interactions we observe a dramatic decrease in axonal and dendritic localization ofthe transporter. Our data suggest that this phenotype is not caused by disrupting the interaction with PICKI (protein interacting with C kinase-1), the so far only PDZ protein known to Interact with DAT, but rather the interaction with yet unidentified PDZ proteins. Instead we hypothesize that PICKI binding regulates DAT electrogenic properties. An in vivo role of C-terminal protein-protein interactions in mediating AMPH action is supported by the ability of a C-terminal membrane permeable DAT peptide to reduce the locomotor response to acute AMPH in mice. We propose therefore i) to determine the role of PDZ interactions in governing axonal and dendritic localization of DAT, and to examine the physiological consequences of disrupting these interactions;11) to investigate the role of PICKI in regulating DAT ion conductances and AMPH-induced dopamine efflux;and iii) to determine the role, in vivo, that binding of CaMKIIa and PICKI to the DAT C- terminus has in establishing the behavioral effects of AMPH. These investigations should contribute significantly to future development of better therapies for drug abuse and perhaps psychiatric disorders. RELEVANCE (See instmctions): The abuse of cocaine and amphetamine causes profound socio-economic problems in the society. However, we know still little about the mechanisms in the brain that are responsible for the development of addiction to these drugs. Insight into the molecular mechanisms governing the activity and availability of their primary target, the dopamine transporter, should prove Instrumental in our search for new therapeutic strategies.